USOO587221 OA United States Patent (19) 11 Patent Number: 5,872,210 Medabalimi (45) Date of Patent: Feb. 16, 1999

54) TRANSFRAME PEPTIDE INHIBITOR OF Yamamoto, Y., et al., “Synthesis of Hexatriacontapeptide WRAL Amide Corresponding to the Proposed Structure of Neu ropeptide Y (NPY)", Peptide Chemistry, 317-322 (1985). 75 Inventor: John L. Medabalimi, Olney, Md. Yamamoto, Y, et al. “Synthesis of Hexatriacontapeptide Amide Corresponding to the Proposed Structure of Neu 73 Assignee: The United States of America as ropeptide Y (NPY)", Chemical Abstracts, vol. 103, abstract represented by the Department of No. 123894 (1985). Health and Human Services, Cason, J., et al. “Identification of Immunogenic Regions of Washington, D.C. the Major Coat Protein of Human Papillomavirus Type 16 that Contain Type-restricted Epitopes' J. Gen. Virol., 21 Appl. No.: 539,432 70:2973–2987 (1989). Cason, J., et al. “Identification of Immunogenic Regions of 22 Filed: Oct. 5, 1995 the Major Coat Protein of Human Papillomavirus Type 16 51) Int. Cl...... A61K 38/04; A61K 39/42; that Contain Type-restricted Epitopes’, Chemical Abstracts, C07K 5/00; CO7H 21/02 vol. 112, abstract No. 116820 (1989). 52 U.S. Cl...... 530/327; 530/328; 530/329; Slootstra, J., et al., "Structural aspects of antibody-antigen 530/330; 424/160.1; 424/208.1; 536/23.1; interaction revealed through Small randam peptide librar 536/23.53 ies”, Molecular Diversity, 1:87–96 (1995). 58 Field of Search ...... 424/160.1, 208.1; Slootstra, J., et al., "Structural aspects of antibody-antigen 530/328, 327, 329, 330; 536/23.1, 23.53 interaction revealed through Small randam peptide librar ies”, Chemical Abstracts, vol. 124, abstract No. 257898. 56) References Cited Robinson, Michael B., et al., “Hydrolysis of the Brain Dipeptide N-Acetyl-L-aspartyl-L-glutamate”, The Jour U.S. PATENT DOCUMENTS nal of Biological Chemistry, 262: 14498–14506 (1987). 5,087,557 2/1992 McClure ...... 435/5 (List continued on next page.) 5,188,950 2/1993 Balani et al. . ... 435/120 5.245,015 9/1993 Fung et al...... 530/388.35 Primary Examiner Jeffrey Stucker 5,342,922 8/1994 Marshall et al...... 530/329 Assistant Examiner Hankyel T. Park Attorney, Agent, or Firm Morgan & Finnegan, L.L.P. FOREIGN PATENT DOCUMENTS 57 ABSTRACT 0 498 784 A2 8/1992 European Pat. Off.. 43 32395 A1 4/1995 Germany. The present invention describes Small, water Soluble pep WO 88/09815 12/1988 WIPO. tides isolated from a native virus inhibitory Sequence that WO 91/13904 9/1991 WIPO. blocks maturation of the virally encoded protease and inhib WO95/27054 10/1995 WIPO. its the mature protease as well. The peptides may be used in OTHER PUBLICATIONS the treatment of Virally infected cells, in the preparation of vaccine formulations, in the generation of clinically relevant Szewczuk, A., et al., “Specificity of Y-Glutamyl Cyclotrans antibodies and anti-idiotypic antibodies and in the genera ferase”, Can. J. Biochem., 53:706-712 (1975). tion of a Screening assay or kit that can be used to identify Szewczuk, A., et al., “Specificity of Y-Glutamyl Cyclotrans other Similarly acting protease inhibitors. ferase', Chemical Abstracts, vol. 83, abstract No. 92952 (1975). 10 Claims, 3 Drawing Sheets

C A A

...& aga Cag gct aat fri Arg Gln Ala Asn Phe Leu Gly Lys 5'-AAU UUU UUA G G G A A G A G A-3 aga cag gCt aat ttt tta agg gaa gat ctg. gicc titc cta Arg Glin Ala Asn Phe Leu Arg Glu Asp Leu Ala Phe Leu 55 8 9

Gag-Pol p 17 P2424 p7 -- TFp6 5,872,210 Page 2

OTHER PUBLICATIONS Zybarth, Gabrielle, et al. “Proteolytic Activity of Novel Human Immunodeficiency Virus Type 1 Proteinase Proteins Anderson, D.C., et al. “Exact Definition of Species-Specific and Cross-Reactive Epitopes of the 65-kilodalton Protein of from a Precursor with a Blocking Mutation at the N Termi Mycobacterium leprae. Using Synthetic Peptides”, The Jour nus of the PR Domain”, Journal of Virology, 68:240–250 nal of Immunology, 141:607-613 (1988). (1994). Battle, J.K., et al. “Immunological Characterization of the Louis, John M., et al. "Kinetics and mechanism of autopro gag Gene Products of Bovine Immunodeficiency Virus,” cessing of human immunodeficiency virus type 1 protease Journal of Virology, 66:6868-6877 (1992). from an analog of the Gag-Pol polyprotein', Proc. Natl Zybarth, G., et al., “Domains Upstream of the Protease (PR) Acad. Sci., 9:7970–7974 (1994). in Human Immunodeficiency Virus Type 1 Gag-Pol Influ Wondrak, Ewald M., et al. “Removal of Zinc is Required for ence PR Autoprocessing”, Journal of Virology, Processing of the Mature Nucleocapsid Protein of Human 69:3878-3884 (1995). Immunodeficiency Virus, Type 1, by the Viral Protease”, The Giam, Chou-Zen, et al. “In Vivo and in Vitro Autoprocess Journal of Biological Chemistry, 269: 21948-21950 (1994). ing of Human Immunodeficiency Virus Protease Expressed Miller, Michael D., et al. “Advances in Automated Docking in Escherichia coli,” The Journal Biological Chemistry, 263: Applied to Human Immunodeficiency Virus Type 1 Pro 14617–14620 (1988). tease”, Methods in Enzymology, 241: 354-70 (1994). Strickler, James E., et al. “Characterization and Autopro Ringe, D., "X-Ray Structures of Retroviral and cessing of Precursor and Mature Forms of Human Immu Their Inhibitor-Bound Complexes”, Methods in Enzymol nodeficiency Virus Type (HIV 1) Protease Purified From ogy, 241:157–177 (1994). Escherichia coli'', Proteins. Structure, Function and Genet Vacca, J. P., “Design of Tight-Binding Human Immunode ics, 6:139–154 (1989). ficiency Virus Type 1 Protease Inhibitors’, Methods in Louis, J. L., et al. "Autoprocessing of the HIV-1 protease Enzymology, 241:311-334 (1994). using purified wild-type and mutated fusion proteins Kempf, D.J., “Design of Symmetry-Based, Peptidomimetic expressed at high levels in Escherichia coli'', Eur: J. Bio Inhibitors of Human Immunodeficiency Virus Protease”, chem. 199,361-369 (1991). Methods in Enzymology, 241:334–354 (1994). Reil, H., et al. “A Heptanucleotide Sequence Mediates Balani, S.K., et al. “Metabolism of L-689,502 by Rat Liver Ribosomal Frameshifting in Mammalian Cells”, Journal of Slices to Potent HIV-1 Protease Inhibitors", Drugs Metabo Virology, 67:5579–5584, (1993). lism and Disposition, 23:185-189 (1995).

U.S. Patent Feb. 16, 1999 Sheet 2 of 3 5,872.210

O O cy CN S-W'ee 101

Z "S) U.S. Patent Feb. 16, 1999 Sheet 3 of 3 5,872.210

FIG. 3

: 5,872,210 1 2 TRANSFRAME PEPTIDE INHIBITOR OF The Gag and Gag-Pol cleavage Sites are a prime target for WRAL PROTEASE protease inhibitors. For example, peptide-based HIV pro tease maturation inhibitors have been generated that target FIELD OF INVENTION these sites in order to interfere with HIV processing The present invention relates to the field of inhibition of (Burgess, K. and Pal, B. (1994), Bioorganic & Med Chem. Viral infectivity by interrupting protease maturation and 2:23-26). Gag and Gag-Pol cleavage Site Sequences have processing, as well as inhibiting of mature protease enzy also been modified to generate peptide-analog inhibitors matic activity. (Marshall, G. R. et al., U.S. Pat. No. 5,342,922; Burgess, K. and Pal, B. (1994), Bioorganic & Med. Chem. 2:23–26). In BACKGROUND OF THE INVENTION addition, the p7/p6 junction of Gag has been identified as a Inhibition of proteases is an increasingly important potential target for protease inhibitors (Billich, S. et al. approach in the control of pathogenic organisms, including (1988), J. Biol. Chem. 263:17905–17908; Roberts, N.A., et retroviruses. Such approaches may also be important in the al. (1990), Science 248:358-361). However, to date, very preparation, processing, and maintenance of various biologi little attention has been given to the Structural or functional 15 significance of the transframe (“TF) region of the viral cal materials, vaccine Stability and cold-chain independent polyprotein. transport may be facilitated by Such methods. Substrate competition is another approach for protease Retroviruses produce a polycistronic mRNA that encodes inhibition; target peptides corresponding to protease active precursor molecules for the Structural and functional viral proteins. A virally encoded aspartic acid protease is respon Site Substrates compete for access to the mature Sible for the processing of the polyprotein precursors Gag recognition Site. In addition, peptides and peptide analogs and Gag-Pol into the mature Structural and replication which mimic Substrate intermediates of enzyme-catalyzed . A gag-pol polyprotein homodimer forms to gen hydrolytic reactions have been described as aspartyl pro erate the Protease catalytic . Protease is then tease inhibitors (Rich, D. L. (1986) Proteinase Inhibitors, released from the precursor in an autocatalytic process. This (Barrett and Salvesson, Eds.) Elsevier Science Publishers 25 BV). Furthermore, such peptide inhibitors have been altered processing is a critical Step in the life cycle of retroviruses, preventing hydrolysis of the Substrate peptide bonds, including human immunodeficiency virus (“HIV”), the etio thereby blocking the protease from releasing the Substrate logical agent of acquired immunodeficiency Syndrome peptide and hydrolyzing the true target Sequence. For (“AIDS”). Improper processing, premature activation of the example, such peptides have been used to inhibit HIV virus protease, or partial inhibition of the enzymatic activity activity and to inhibit the proliferation of HIV in infected during viral replication leads to defects in Viral assembly and human lymphocytes (Voges, K. P., et al., U.S. Pat. No. the formation of non-infectious, aberrant virus particles. 5,145,951). A limitation to this approach is that the inhibitor The HIV protease is autocatalytic, releasing itself from peptide may still be released from its complex with the the precursor molecule by cleavage at two Sites in the protease and again become available for processing of the precursor polyprotein open reading frame (“ORF); the 35 true target Sequence. amino (“N-”) terminal extension of the Protease is removed, Poor water solubility of protease inhibitors further impairs followed by cleavage at the carboxyl (“C-”) terminus the in vivo utility of HIV peptide-based protease inhibitors (Strickler et al. (1989), Proteins 6:139–154). Analysis of (Robins, T. and Plattner, J. (1993), J. AIDS 6:162–170), and Viral mutants Suggested that the N- and C- cleavages are has been addressed by the addition of Solubilizing groups interdependent (Louis, J. M. (1991), Euro. J. Biochem. 40 Such as poly-lysine, arginine methyl ester, glutamic acids, 199:361-369; Louis, J. M., et al. (1991), Adv. Exp. Med. aspartic acids, Asp-Arg, Gly-Lys-Lys and dextran in order to Biol. 306:499–502). In addition, autoprocessing at the C improve the solubility of some inhibitory compounds cleavage Site is not significantly affected by the presence of (Toniolo, C., et al. (1994), J. Med. Chem. 37:4558–4562; the N-terminal precursor sequence. (Valverde et al. (1992), Hostetler, K. Y., et al. (1994), Biochem Pharmacol J. Gen. Virology 73:639-651). 45 48: 1399–1404). The present invention overcomes such limi A two Step mechanism for autoprocessing of the HIV tations without requiring chemical modification Strategies. protease precursor polyprotein has been proposed (Louis, J. Thus, present day approaches cannot completely block M., et al. (1994), Proc. Natl. Acad. Sci. USA, Virus activity or the resulting virus-induced pathology. Pre 91:7970-7974), wherein a first step is N-terminal Protease vious reports fail to identify a useful mechanism for inhi cleavage, releasing an active protease-polymerase 50 bition and control of HIV protease maturation as disclosed intermediate(s), followed by a Second step of release of in the present invention. mature Protease enzyme by cleavage at the C-terminus. The Furthermore, up to the present, no Satisfactory treatment HIV protease autoprocessing Suggests that HIV regulates has been available which is based solely on inhibition of the protease expression in order to prevent premature complete mature viral protease. Current approaches to protease inhi cleavage (Arrigo, S.J., et al. (1995), DNA and Cell Biology, 55 bition rely on peptides that are hydrophobic and which are 14:15-23). It has also been noted that deletion of one region directed to specific Sequences of the active Site in order to of the polyprotein precursor, the p6 region, enhances pro competitively inhibit protease activity. Such approaches are cessing. (Partin, K. et al. (1991), Proc. Natl. Acad. Sci. USA reported to result in the rapid Selection of Viral variants that 88(11):4776–4780). are resistant to such inhibitors (Winslow, D. L. and Otto, M. Proper processing at HIV Gag and Gag-Pol cleavage Sites 60 J. (1995), AIDS9 (suppl A):S183-S192). is crucial for viral infectivity (Robins and Plattner, (1993).J. It is therefore an object of the present invention to develop AIDS 6:162-170). Cleavage sites in Gag-Pol include inter a method of inhibition of a virus encoded protease by alia: inhibiting the maturation of the enzyme. Another object of p17/p24/p2/p7/p1/p6/p51/p15/p34. (See Table I, and see the present invention relates to isolated forms of naturally FIG. 1; see also Table I, Methods in Enzymology, 241:265, 65 occurring, Virally encoded inhibitory peptide Sequences (Eds. L. C. Kuo and J. A. Shafer); Dunn, B.M., et al. (1978), which regulate protease maturation, arrest activation and/or J. Biol. Chem. 253:7269–7276). reduce catalytic activity of the protease. 5,872,210 3 4 It is a further object of the present invention to combine idiotype antibodies of TF peptide inhibitors; and a kit for competitive inhibition of protease activity with inhibition of Screening compounds for use as aspartyl protease inhibitors protease maturation and therefore inhibition of activation of using the peptide inhibitors and an aspartyl protease as a the protease itself. This approach will avert the rapid Selec Substrate. tion of viral variants that are resistant to the inhibitor. The present invention is the first disclosure of TF peptides Another object of the present invention relates to use the as inhibitors of protease maturation and of protease enzyme inhibitory peptides in a Screening assay System of test activity. The peptides described by the present invention are compounds wherein additional, potent as partial protease short and highly water soluble. inhibitors may be identified that also block protease activity According to the present invention, the TF peptide inhibi either by competitive inhibition or by inhibition of protease tor comprises a peptide with about 2 to 20 amino acids maturation. arranged in a specific Sequence which is useful for the Because the natural inhibitory region is important in the inhibition of HIV protease by blocking maturation of the control and regulation of the Virus life cycle, it is a further protease and by competitive inhibition of the enzymatic object of the present invention to use isolated peptides activity of the mature protease. derived from the inhibitory region in a formulation to 15 Stabilize a virus preparation, for example, as part of a BRIEF DESCRIPTION OF THE DRAWING Stablilized vaccine preparation, by a method of inhibition of protease induced degradation or processing of retroviral FIG. 1. Sequence surrounding the frame-shift site in virions. The instant TF peptide inhibitors are further pack HIV-1: The Gag and Pol polyproteins of HIV-1 are indicated aged in the virus particle to maintain the protease and block by bolded lines. Short arrows indicate the various sites of autolysis of the protease for function early during virus cleavage by the viral protease. The DNA and protein infection. Sequences Spanning transframe cleavage junctions in the Yet another object of the present invention is to use Gag-Pol polyprotein are shown. The RNA sequence Span antibodies and anti-idiotypic antibodies against the inhibitor ning the conserved signal sequence 6UA (underlined) and element to inhibit virion maturation and for direct treatment 25 the secondary structure is shown. TFP and TFp6 denote the of virus-infected cells. eight TF peptide residues flanking the Gagp7 (Phe 1 to Phe8) and remainder of the transframe region residues Phe9 to the SUMMARY OF THE INVENTION N-terminus of the protease, respectively. The DNA and The present invention relates to an inhibitor of a crucial corresponding protein Sequence of the Pol open reading event in the viral life cycle, which functions by targeting frame are shown in bolded letters. Gag p1 amino acids are maturation of a viral protease. The inhibitors of the present italicized. invention comprise an isolated portion of the transframe FIG. 2. Lineweaver-Burk plots of HIV-1 protease cata region of the retroviral precursor polyprotein and is referred lyzed hydrolysis of Substrate 1 at various concentrations of to herein as the transframe or “TF region. The inhibitors of the inhibitor tripeptide, Glu-Asp-Leu referred to herein as the present invention are novel antiviral agents that block 35 “EDL" in 50 mM sodium formate at pH 4.25 containing 150 polyprotein maturation as well as competitively inhibiting mM of HIV-1 protease, 2.5 mM dithiothreotol (“DTT) at the enzymatic activity of the mature protease. In addition, 25 C. Inset is a plot of the apparent K. versus inhibitor inhibition of protease maturation avoids the rapid Selection concentration. of Viral variant resistance mutants. FIG. 3. A plot of K, for HIV-1 protease-catalyzed hydroly The transframe region encompasses a Sequence that is 40 sis of substrate 1 vs. pH. K. was obtained from a plot of 1/V released prior to the release of the fully active protease from versus inhibitor concentration (referred to herein as “I”) at the precursor polyprotein. It is a region wherein a reading a saturating concentration of Substrate 1 (390 uM). Reac frameshift occurs during translation of the polyprotein pre tions were carried out between pH values of 3 to 5 in 50 mM cursor (see FIG. 1). The inhibitors of the present invention Sodium formate or 100 mM Sodium acetate buffers contain comprise a peptide which is inhibitory of retroviral matu 45 ing 150 mM of HIV-1 protease, 2.5 mM DTT at 25° C. ration and enzymatic activity. Examples of Such peptides comprise SEQ ID NO: 19 and fragments thereof having DETAILED DESCRIPTION OF THE protease inhibitory activity. Additional inhibitory peptides INVENTION include, SEQ ID NOS: 1-18. 50 The present invention relates to the identification of a The Scope of the present invention also relates to a method transframe (“TF) region within a retroviral polyprotein for treating HIV infections in a human comprising admin containing a novel TF peptide inhibitor of retroviral pro istering thereto an effective amount of at least one TF tease. This inhibitor appears to play a regulatory role in the peptide inhibitor, or a therapeutically acceptable Salt thereof. retroviral life-cycle by modulating the Synthesis of active Also included within the Scope of the present invention is 55 retroviral protease. The TF peptide inhibitors of the present a method for protecting human cells against HIV pathogen invention function as inhibitors of retroviral protease matu esis comprising treating Said cells with an anti-HIV effective ration. In addition, the TF peptide inhibitors act as competi amount of at least one TF peptide inhibitor compound, or a tive inhibitors of enzymatic activity of the functional, therapeutically acceptable Salt thereof. mature viral protease. The present invention encompasses TF inhibitory pep 60 The TF peptide inhibitors of the present invention may be tides, pharmaceutical compositions containing the inhibitory functional in many retroviral Systems to curb the spread of peptides, methods of using the peptides to inhibit maturation infective virus. In particular, the spread of human immuno and activity of the HIV-1 protease in treatment and preven deficiency viruses (known as “HIV") such as HIV-1 and tion of HIV-induced pathogenesis, methods for Screening HIV-2 may be inhibited by a TF peptide inhibitor. Other test compounds (peptide or non-peptide) for use as HIV 65 retroviruses which may be inhibited by the TF peptide protease inhibitors using a complex of TF peptide inhibitor inhibitors of the present invention include, but are not and the viral protease as a Substrate; antibodies and anti limited to, HLV-I, HTLV-II, BLV, EIAV, FIV, SIV, STLV and 5,872,210 S 6 Visna virus. The TF peptide inhibitors of the present inven and excision of the TF inhibitor from the precursor polypro tion also inhibit other mammalian aspartyl proteases. tein. The mature, fully active protease is then released from The TF peptide inhibitor region of the present invention the polyprotein by a Subsequent intramolecular cleavage at comprises the transframe region, located within the Gag-Pol its N-terminus and a intermolecular cleavage at its polyprotein precursor. In particular, the “transframe” or “TF 5 C-terminus. This mature protease then hydrolyses other region' encompasses a region wherein a translational frame cleavage Site peptide bonds in the Gag and Pol domains to shift occurs. TF peptide inhibitor sequences exist within this release the necessary mature enzymes and Structural proteins region and comprise a highly conserved Sequence. One for virus maturation, unless a TF inhibitor is present, in preferred TF region encompasses a short region of p7, and which case protease enzymatic activity is inhibited in trans. four amino acids at either end flanking the N- and C-termini Additionally, a TF peptide inhibitor of the present inven of TFP6 and p7 respectively (see FIG. 1, and Table 1). tion also may trap and maintain the protease in an inactive Another preferred region comprises SEQ ID NO: 19 or form in the cores of the infective virions, in cis or trans, fragments thereof, wherein the fragments have aspartyl thereby blocking autolysis. protease inhibitory activity. "Fragments', as the term is used The inhibitory activity of the TF peptides is pH depen herein comprises Sequences which are shorter than the 15 dent. For example, inhibition of the HIV protease by TF named Sequence and have the characteristics of being inhibi peptide inhibitors is optimal between pH 3-7 (see FIG. 3). tory of aspartyl protease activity. Fragments of a particular In one embodiment of the present invention, the TF Sequence can vary in length and in fact, may be as short as peptide inhibitor comprises a 16 amino acid peptide, 2 amino acids in length. Fragments may further carry “RQANFLREDLAFLQGK” (SEQ ID NO: 19) encoded modified amino acids, peptide bonds or end groups. within the N-terminal amino acid Segment of the transframe A "peptide,” as the term is used herein, is a Sequence of region of HIV-1, or fragments of SEQ ID NO: 19 which amino acids linked together in a chain by peptide bonds. The have aspartyl protease inhibitory activity. term "peptide' includes peptide analogs, wherein one or In another embodiment of the present invention the TF more amino acid Side chains, reactive groups, peptide bonds, peptide inhibitor comprises a specific eight amino acid or other moieties have been altered either through chemical 25 peptide, “FLREDLAF" (SEQ ID NO: 1) encoded within the modifications or by Some other process. In producing the TF N-terminal amino acid Segment of the transframe region of peptide inhibitors of the present invention, it may be desir HIV-1. able to modify the peptides, the amino acids or the peptide In yet another embodiment of the present invention a linkages in one or more positions of the peptide Sequence. specific three amino acid peptide, EDL (SEQ ID NO: 4) For example, it may be advantageous to alter the peptide provides potent inhibitory activity. The EDL peptide bond to render it non-hydrolyzable. It will be understood by strongly and selectively inhibits the HIV aspartic acid those skilled in the art that such modifications may be protease, as demonstrated by its inability to inhibit , a achieved by standard procedures (Winslow, D. L. and Otto, mammalian aspartic acid protease. Further, the EDL peptide M. J. (1995), AIDS 9 (suppl A):S183-S192; Meek, T. D. is highly Soluble in water, in contrast to previously described (1992), J. Enzym. Inhib. 6:65-98; Vaillancourt, M. et al. 35 peptide inhibitors. In addition, binding of a TF peptide (1994) Bioorg. Med. Chem, 2:343–355). It will further be inhibitor to the HIV protease is not dependent on a high salt understood by those skilled in the art that other modifica concentration. tions capable of blocking protease cleavage can be used to Smaller TF peptide inhibitor compositions include, but achieve inhibition. 40 are not limited to FLREDLAF (SEQ ID NO 1), GEDLAF The term “pharmaceutically acceptable carrier as used (SEQ ID NO2), EDLA (SEQ ID NO3), EDL (SEQ ID NO herein means a non-toxic, generally inert vehicle for the 4), RED (SEQ ID NO 5), DLAF (SEQ ID NO 6), DEL(SEQ active ingredient, which does not adversely affect the ingre ID NO 7), EEL (SEQID NO 8), DDL (SEQ ID NO 9), ENL dient. (SEQ ID NO 10), EKF (SEQID NO 11), EDF (SEQID NO The term “effective amount” as used herein means a 45 12), DD (SEQ ID NO 15), DE (SEQ ID NO 16), DQ (SEQ predetermined amount of the peptides of this invention ID NO 17), DDF (SEQ ID NO 13), or FDDF (SEQ ID NO Sufficient to be effective against a retroviral infection, Such 14). (See Table 2). For example, the three amino acid as HIV in a human. An effective amount may also refer to peptide, EDL (“the trimer”) is a short, water soluble peptide an amount Sufficient to inhibit aspartyl protease activity. with highly potent protease inhibitory character. In addition to inhibiting the enzymatic activity of the 50 The TF peptide inhibitor can be synthesized in a number protease, the TF peptide inhibitors of the present invention of ways. The peptides can be isolated from Viral Source play a regulatory role in the HIV lifecycle. During the HIV material. The peptides may also be chemically Synthesized, lifecycle, two HIV polyprotein precursors, Gag and Gag-Pol free of any HIV virus, reducing risk of exposure to virus to are cleaved by the HIV-encoded protease to yield mature Zero. Chemical Synthesis ensures a high degree of homoge Viral Structural and functional proteins, including the pro 55 neity under controlled conditions and permits chemical tease itself. The mature protease exhibits 300-600 fold modifications to be incorporated along the way (Merrifield, greater proteolytic activity than precursor protease con R. B. (1963), J. Amer: Soc., 85:2149). tained within the Gag-Pol precursor. Even partial inhibition Alternatively, the peptides may be produced by recombi of protease activity interferes with the HIV lifecycle and nant DNA techniques using methods Such as those in leads to defects in Viral assembly including the production of 60 Sambrook, 1988, Molecular Cloning. A Laboratory Manual non-infectious virion particles. The Gag-Pol precursor (2nd Edition) that are well understood in the art. polyprotein containing both the TF peptide inhibitor region The TF peptide inhibitors may be modified by techniques and a protease regulates the HIV lifecycle by maintaining understood in the art (Winslow, D. L. and Otto, M.J. (1995), the protease in a relatively inactive form with low enzymatic AIDS 9 (suppl A) :S183-S192; Meek, T. D. (1992), J. activity at neutral pH, called cis regulation, until the 65 Enzym. Inhib. 6: 65-98; Rich, D. L. (1986), Proteinase polyproteins are assembled into immature particles. At that inhibitors, (Barrett and Salvessen, Eds.) Elsevier Science point, reduction of pH (optimal at pH 5.0) results in cleavage Publications, BV), substituting one or more amino acid 5,872,210 7 8 Side-chain groups or other groups or bonds or linkages in which is determined by the solubility and chemical nature of order to improve the inhibitory character of the peptide. For the peptide, chosen route of administration and Standard example, alteration of the peptide bond in order to render the biological practice. For oral administration, the peptides or peptide non-hydrolyzable, by the “NOR' method described a therapeutically acceptable Salt thereof can be formulated in by Burgess, K. and Pal, B. (1994), Bioorganic & Med. unit dosage forms Such as capsules or tablets each contain Chem. 2:23-26) in which the “Nor” peptide analog inhibi ing a predetermined amount of the active ingredient, ranging torS eXclude the natural cleavage/active sites. Other modi from about 25 to 500 mg, in a pharmaceutically acceptable fications may include aldehydes (Sarubbi, E., et al. (1993) carrier. For topical administration, the peptides can be FEBS Lett., 319:253–256) or “Mirror” D-amino acids which formulated in a pharmaceutically acceptable vehicle con act as irreversible inhibitors blocking hydrolysis. The main taining 0.1 to 10 percent, preferably 0.5 to 5 percent, of the tenance of the three dimensional conformation, physical active agent. Such formulations can be in the form of a constraints or molecular interactions may preserve the cream, lotion, Sublingual tablet, or preferably a transdermal inhibitory quality of the TF peptide inhibitors found in the patch or buccal patch. virion. Furthermore, the TF peptide inhibitors can be used to The pharmaceutical composition of the present invention obtain crystallographic information regarding the three 15 can take the form of a lyophilized powder of the active dimensional Structure of the TF peptides alone or complexed Substance, to be dissolved immediately before use in a with the protease. This information may Suggest new inhibi physiological Solution for the purpose of injection. For tors through molecular modelling techniques as known in parenteral administration, the peptide of the formula is administered by either intravenous, Subcutaneous or intra the art. muscular injection, in compositions with pharmaceutically The ability of TF peptides to inhibit aspartyl proteases, acceptable vehicles or carriers. For administration by Such as HIV aspartyl protease is significant. Use of Such injection, it is preferred to use the peptide in Solution in a peptides in a pharmaceutical composition allows for the Sterile aqueous vehicle which may also contain other Solutes control and regulation of HIV protease in vitro and in Such as buffers or preservatives as well as Sufficient quan humans infected with the HIV virus. TF peptide inhibitors tities of pharmaceutically acceptable Salts or of glucose to can therefore be used to control and regulate HIV virus 25 make the Solution isotonic. The pharmaceutical composition activity in vitro and in vivo. It is recognized that inhibition according to the invention can also take a form which is of the HIV protease has important implications for control of Suitable for oral administration. For example, Suitable forms HIV infection in humans. are tablets, food gelatin capsules, dragées, powders and In one preferred embodiment, the TF peptides are used to granules. The formation of Such oral forms is well-known to inhibit HIV activity in a population of cells. For example, those skilled in the art. Any of the known formulations are the TF peptide inhibitor may be used to inhibit virus activity useful in preparing the instant oral pharmaceutical compo in a retrovirally infected human. One or more peptides may Sitions. be used to treat, for example, an HIV infected patient. The present invention also covers the use of TF peptide The peptides of this invention can be obtained in the form 35 inhibitor compositions in combination with other medicinal of a therapeutically acceptable Salt. In the instance where a compositions intended for the treatment of retroviral infec particular peptide has a residue which functions as a base, tions and tumors. Immunostimulants and immunomodula examples of Such Salts are those with organic acids, e.g. torS Such as for example cytokines, including IL-2, IL-12 acetic, lactic, Succinic, benzoic, Salicylic, methaneSulfonic and interferon molecules can be used in combination with or p-toluenesulfonic acid, as well as polymeric acids Such as 40 the present invention. tannic acid or carboxymethyl cellulose, and also Salts with Suitable vehicles or carriers for the above noted formu inorganic acids Such as hydrothalic acids, e.g. hydrochloric lations can be found in Standard pharmaceutical texts, e.g. in acid, or Sulfuric acid, or phosphoric acid. If desired, a “Remington's Pharmaceutical Sciences”, 16th ed. Mack particular acid addition Salt is converted into another acid Publishing Company, Easton, Pa., 1980. addition Salt, Such as a non-toxic, pharmaceutically accept 45 The dosage of the peptide will vary with the form of able Salt, by treatment with the appropriate ion exchange administration, the frequency of administration, the nature resin in the manner described by Boissonnas, R. A., et al. and Severity of the infection and the particular active peptide ((1960), Helv. Chim. Acta, 43:1849). agent chosen. Furthermore, it will vary with the particular In the instance where a particular peptide has one or more host under treatment. Generally, treatment is initiated with free carboxyl groups, examples of Such Salts are those with 50 Small dosages Substantially less than the optimum dose of the Sodium, potassium or calcium cations, or with Strong the peptide. Thereafter, the dosage is increased by Small organic base S, for example, trie thylamine or increments until the optimum effect under the circumstances N-methylmorpholine. is reached. The actual dosage administered will be deter In general, the therapeutically acceptable Salts of the mined by physiological factorS Such as age, body weight, peptides are biologically fully equivalent to the peptides 55 Severity of condition and/or chemical history of the patent. themselves. With these considerations in mind, the dosage of the TF The cell protective effect of the peptides or their thera peptide inhibitor composition for a particular Subject can be peutically acceptable Salts can be demonstrated by micro readily determined by the physician. In general, the peptide biological procedures for evaluating the effect of test com is most desirably administered at a concentration level that pounds in inhibiting the cytopathogenicity of HIV in human 60 will generally afford antivirally effective results without CD4" cell types; for example, see Baba, M., et al. (1987), causing harmful or deleterious Side effects. However, it is Biochem. Biophys, Res. Comm., 142:128). noted that in extreme cases a dosage approaching the toxic When peptides of the instant invention, or a therapeuti level may be an acceptable treatment protocol. cally acceptable salt thereof, is used to combat HIV infec For oral administration, the peptide or a therapeutically tions in a human, Such peptides can be administered orally, 65 acceptable Salt is administered in the range of 0.01 to 75 mg topically or parenterally, in a vehicle comprising one or per kilogram of body weight per day, with a preferred range more pharmaceutically acceptable carriers, the proportion of of 2.5 to 20 mg per kilogram. 5,872,210 9 10 With reference to systemic administration, the peptide of Exemplary antibody molecules for use in the detection the formula is administered at a dosage of 10 ug to 1000 mg methods of the present invention are intact immunoglobulin per kilogram of body weight per day, although the afore molecules, Substantially intact immunoglobulin molecules mentioned variations will occur. However, a dosage level or those portions of an immunoglobulin molecule that that is in the range of from about 50 lug to 500 mg per contain the antigen , including those portions of kilogram of body weight per day is most desirably employed immunoglobulin molecules known in the art as F(ab), F(ab'), in order to achieve effective results. F(ab') and F(v). Polyclonal or monoclonal antibodies may Although the formulations disclosed hereinabove are be produced by methods known in the art (Kohler and effective and relatively safe medications for treating HIV Milstein (1975), Nature 256:495-497; Campbell (1985), infections, the possible concurrent administration of these Laboratory Techniques in Biochemistry and Molecular formulations with other antiviral medications or agents to Biology, Vol. 13, Burdon, et al. (Eds.), Elsevier Science obtain beneficial results is not excluded. Such other antiviral Publishers, Amsterdam). The antibodies or antigen binding medications or agents include Soluble CD4, thalidomide, fragments may also be produced by genetic engineering. The dide oxy inoSine, dide oxythy mine, Zidovudine, technology for expression of both heavy and light chain dideoxycytidine, gancyclovir, acyclovir, phosphonoformate, 15 genes in E. coli is the Subject of the PCT patent applications: ama tradine, ribavarin, antiviral interferons (e.g. publication number WO 901443, WO 901443, and WO C-interferon, C.-interferon, or interleukin-2) or aerosol 9014424 and in Huse et al. (1989), Science 246:1275–1281. pentamidine, and other Substances used in anti-HIV therapy The present invention is particularly advantageous in the in particular, the TF peptide inhibitor formulations may be control of virus infection in that the TF peptide inhibitors used in conjunction with other protease inhibitorS Such as combine inhibition of protease maturation with a powerful those described in Winslow, D. L. and Otto, M. J. (1995), competitive inhibitory activity against mature protease. AIDS 9 (supp A):S183-S192). Indeed, this two-fold activity is a substantial advance. With Treatment of infected cells to inhibit virus activity may be the TF peptides inhibitors of the present invention, mature for a Specific period of time or may be continuous. Virus protease production is dramatically reduced, and any avail activity may be measured by monitoring levels of Viral 25 able mature or nascent protease is Secondarily inhibited by protein, Such as P24, or by measuring reverse transcriptase a competitive mechanism. levels, or by monitoring virus protein activity by S-met Yet another embodiment of the present invention relates pulse-chase labelling and immunoprecipitation experiments, to use of the peptides to identify new compounds, Such as or by other methods which are well known by one of skill peptides or non-peptides with even greater anti-viral prop in the art. (Kayeyama, S., et al. (1994), AIDS Res. and erties for inhibiting protease activity and/or maturation in a Human Retroviruses, 10:735-745). Screening assay. A test Screening kit, relying on the well Another embodiment of the present invention relates to understood methods of competitive inhibition has been the generation and use of antibodies directed against the TF developed based on the TF peptides. Such a kit comprises a peptide inhibitors. Such antibodies have many uses. The protease and at least one TF peptide inhibitor, which forms anti-TF peptide inhibitor antibodies can be used to inhibit 35 a complex, Said complex or protease being bound to a Solid virus activity directly by binding to the TF peptide inhibitor Support, Such as a magnetic bead or plastic matrix. In Some Sequence, thereby blocking cleavage and release of the cases, it may be preferred that the peptide(s) are labelled. mature protease. Also, the anti-TF peptide inhibitor antibod Labelling agents are well-known in the art. For example, ies can be used to generate anti-idiotypic antibodies that can labelling agents include but are not limited to radioactivity, directly interfere with protease activity itself. Anti-TF pep 40 chemiluminescence, bioluminescence, luminescence, or tide inhibitor antibodies and any anti-idiotypic antibodies, other identifying "tags' for convenient analysis. may be used as “intracellular vaccines' in treating virally Varying amounts of test compounds, Such as peptides or infected cells. Such intracellular uses involve the use of non-peptide compounds are added to the complex in order to So-called Single chain antibodies which can be introduced displace of the original peptide complexes. Analysis of the into cells by methods well-known in the art (Huston, J. S., 45 release of the peptides, by, for example, monitoring the et al. (1988), Proc. Natl. Acd. Sci., USA) or may be directly identifying tag or label following incubation with the test expressed within cells under the appropriate cellular con Sample, indicates the relative binding affinity of the test trols. Sample in the complex and thus is indicative of identification The present invention further relates to antibodies and of a new anti-Viral compound. The new complex may then anti-idiotype antibodies generated from TF peptide inhibitor, 50 be assayed for inhibition of protease activity by Standard (also referred to as “TF antibodies”). In this embodiment of kinetic assays (see Example 2 below; Ferhst, A. (1977), the invention, TF antibodies are monoclonal or polyclonal in Enzyme Structure and Mechanisms, W. H. Freeman and Co., origin. The TF peptide inhibitors used to generate the TF San Francisco; Segal, I. H. (1975), , John antibodies may be from natural or recombinant Sources or Wiley and Sons, New York). generated by chemical synthesis. Natural TF inhibitor pep 55 Solid matrices are available to the skilled artisan. Solid tides can be isolated from viral samples. Synthetic TF phases useful as matrices for the present invention include inhibitor peptides may be custom ordered or commercially but are not limited to polystyrene, polyethylene, made based on the amino acid Sequences of the present polypropylene, polycarbonate, or any Solid plastic material invention or chemically Synthesized by methods known to in the shape of test tubes, beads, microparticles, dip-sticks, one skilled in the art (Merrifield, R. B. (1963), J. Amer: Soc., 60 plates or the like. Additionally matrices include, but are not 85:2149). If the peptide is too short to be antigenic it may be limited to membranes, 96-well microtiter plates, test tubes conjugated to a carrier molecule to enhance the antigenicity and Eppendorf tubes. Solid phases also include glass beads, of the peptide. Examples of carrier molecules, include, but glass test tubes and any other appropriate shape made of are not limited to, human albumin, bovine albumin and glass. A functionalized Solid phase Such as plastic or glass keyhole limpet hemo-cyanin (“Basic and Clinical Immunol 65 which has been modified So that the Surface carries carboxyl, ogy” (1991) Stites, D. P. and Terr A. I. (eds) Appleton and amino, hydrazide, or aldehyde groups can also be used. In Lange, Norwalk, Conn., San Mateo, Calif.). general Such matrices comprise any Surface wherein a 5,872,210 11 12 ligand-binding agent can be attached or a Surface which pH-rate profile for the autoprocessing reaction showed that itself provides a ligand attachment site. cleavage of the N-terminal TFp6 to release the mature The test compound may be any peptide or non-peptide Protease is dependent on the ionization of groups with pK's composition in a purified or non-purified form. Chemical of 4.9 and 5.1 (see FIG. 3). compounds, Synthetic compounds, biological compounds or other specimens may be used from any Source, including Autoprocessing of ATFP-TFp6-PR precursor to produce plant and animal. The test compound may also comprise a the mature Protease occurs in two steps. In the first Step, the complex mixture or “cocktail” of molecules. Protease hydrolyses the peptide bond corresponding to the Another embodiment of the present invention involves a ATFP-TFp6 junction to produce the enzyme intermediate, method for Stabilizing virus preparations. Virus degradation TFp6-PR. The TFp6-PR which possesses low intrinsic cata is delayed at low temperatures and vaccine preparations lytic activity, comparable to that of A TFP-TFp6-PR is then often require Storage at low temperature (below 0° C.). converted in a Second Step to the mature Protease concomi Treatment of virus preparations, Such as virus-based vaccine tant with a large increase in enzymatic activity. preparations with the TF peptide inhibitors of the present invention serves to reduce or even obviate the need for cold 15 Table 1 Sets forth the cleavage recognition Sequences for Storage of virus or vaccine preparations, by inhibiting the HIV-1 polyprotein cleavage sites. In addition, Table 1 dem proteolytic activity of the protease contained in the viral onstrates the relative efficiency of cleavage at each Site, preparation. based upon K/K.

TABLE 1. Gag Cleavage Sites ka/K, mM's Pol Cleavage Sites kcal/Km. mMs. p17/p24 SQNY* PIVO 45.O p7/TFP EROAN*FLRED 1.9 x 10 p24/p2 ARVLAEAM 9O.O TFP/TFp6 DLAFLOGK 1.2 p2/p7 ATIMMORG 74.O TFp6/PR* SFNF*POIT 7.0 p7/pl ROANFLGK 1.6 PR/RT TLNF*PISP 24.0 p1/p6 PGNFLOSR O6 RT/RN AETF*YVDG 1.O.O RN/IN RKIL*FLDG 2OO.O *PR refers to HIV-1 protease.

For example, a live, attenuated virus-based vaccine, a EXAMPLE 2 killed Virus Vaccine, or a Sub-unit Vaccine preparation may INHIBITION OF HIV-1 PROTEASE BY TF be stabilized in the presence of the protease inhibitors PEPTIDE INHIBITORS 35 described by the present invention. Furthermore, polypro Peptides were at least 98% pure as determined by chro tein or protease preparations may be similarly treated and matography. Stock Solutions of all peptides were prepared in stabilized. water except peptides 1 and 2 (see Table 2) which were All publications, patents and articles referred to herein are dissolved in 50 mM Sodium bicarbonate. The Solution expressly incorporated herein in toto by reference thereto. concentration of peptides containing phenylalanine was The following examples are presented to illustrate the 40 determined spectrophotometrically at 257 nm using the present invention but are in no way to be construed as extinction coefficient for acetylphenylalanine in water as limitations on the Scope of the invention. It will be recog described in CRC Handbook of Biochemistry, Ed. H. A. nized by those skilled in the art that numerous changes and Sober, The Chemical Rubber Co., Cleveland Ohio. Substitutions may be made without departing from the Spirit HIV-1 protease was obtained and its active site concen and purview of the invention. 45 tration was determined at 25 C. Purified enzyme was stored as a 5-8 uM stock solution in 50 mM NaOAc (pH 5.0), 1 EXAMPLE 1. mM DTT, 1 mM EDTA, 0.05% reduced Triton X-100 at REGULATION OF PROTEASE MATURATION -80 C. Porcine pepsin was prepared as 1-2 mg/ml in 50 AND AUTOPROCESSING mM formic acid (pH 3.0). The peptide concentrations were 50 adjusted for their protein content. HIV protease (“PR”) was fused to 48 amino acids of the HIV-1 protease activity was measured at 25 C. using native transframe sequence (“TFp6”) at its N-terminus, substrate I (Lys-Ala-Arg-Val-Nle-Phe (NO)-Glu-Ala-Nle designated TFp6-PR. In another protease fusion, ATFP NH wherein Nile is norleucine and Phe (NO) is TFp6-PR, 6 amino acids of the transframe octapeptide (SEQ 4-nitrophenylalamine). In a typical assay, 2.5 ul of enzyme ID NO: 1) (ATFP; A=truncated) further flanking the TFp6 55 was added to 97.5 ul of buffer and 2.5 mM DTT in the were fused to TFp6-PR. The first-order rate constant for the presence or absence of varying concentrations of inhibitor. autoprocessing of TFp6-PR to release the mature Protease is The reaction was initiated by the addition of 10 ul of a 4 mM identical to previously published results for the autoproceSS solution of substrate I in water and monitored by following ing of the model precursor MBP-ATF-PR-APol providing the decrease in absorption at 310 mM in a 100 ul spectro direct evidence that the intramolecular cleavage that occurs 60 photometric cell. Pepsin was assayed using Substrate II at the N-terminus of Protease is independent of the length of (Phe-Gly-His-Phe(NO-)-Phe-Ala-Phe(OCH). The reaction Sequence flanking the terminus of the Protease. The rate of was monitored by following the decrease in absorbance at disappearance of TFp6-PR and that of the appearance of the 269 nm in a final volume of 110 ul of 50 mM formate buffer, mature Protease and enzymatic activity are identical. The pH 3.0, containing 150 mM and 160 uM enzyme and first order rate constant is identical to that reported for the 65 substrate, respectively, at 25 C. Concentrations of substrate model polyprotein, MBP-ATF-PR-APol (Louis, J. M., et al. Solutions were determined spectrophotometrically at 280 (1994), Proc. Natl. Acad. Sci. USA, 91:7970–7974). The . 5,872,210 13 14 K, was obtained from a plot of the apparent K. VS. I for ID NO: 6) to the enzyme. As shown in Table 3, the K, for peptides 1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2), and 4 (SEQ the peptide, Glu-Asp-Leu, inhibition of HIV-1 protease ID NO: 4) as shown in FIG. 2. For the others, K, was catalyzed hydrolysis of substrate I was 435+65 uM and obtained from a plot of 1/V VS II at a Saturating concen 1001--170 uM in the presence of 1M and 2M NaCl, respec tration of the Substrate. The enzyme was assayed in the tively (50 mM sodium formate, pH 4.0, and 2.5 mM DTT at presence of the inhibitor in 50 mM sodium formate at pH 25 C.). Similarly, the K, for Glu-Asp-Phe and the 4.25 and 2.5 mM DTT. The final enzyme and substrate tetrapeptide, Asp-Leu-Ala-Phe increased to 410+25 uM and concentrations were 150 mM and 390 uM, respectively. 3260+390 uM, respectively, in the presence of 1M NaCl. Peptides 12a and 12b were assayed at pH 4.4 and contained 0.083% and 0.02% DMSO, respectively. ND indicates no 10 TABLE 3 inhibition tested up to a concentration of 50 mM peptide. The octapeptide (SEQ ID NO: 1) and shorter peptides are PEPTIDE NaCl Concentration, M K. M competitive inhibitors for HIV-1 protease-catalyzed Glu-Asp-Leu O 52 - 10 hydrolysis of substrate I as shown in FIG. 2. The measured (SEQ ID NO: 4) 1. 435 65 K.'s are provide in Table 2.

TABLE 2 PEPTIDE K, itM 1. Phe-Leu-Arg-Glu-Asp-Leu-Ala-Phe (SEQ ID NO: 1) 98 10 2 Gly-Glu-Asp-Leu-Ala-Phe (SEQ ID NO: 2) 64 13 3 Glu-Asp-Leu-Ala (SEQ ID NO:3) 16O20 4 Glu-Asp-Leu (SEQ ID NO: 4) SO 9 5 Arg-Glu-Asp (SEQ ID NO: 5) 3360 + 400 6 Asp-Leu-Ala-Phe (SEQ ID NO: 6) 27O 25 7 Asp-Glu-Leu (SEQ ID NO: 7) 230 20 8 Glu-Glu-Leu (SEQ ID NO:8) 31O 25 9 Asp-Asp-Leu (SEQ ID NO: 9) 140 - 6 1O Glu-Asn-Leu (SEQ ID NO: 10) 3070 + 210 11 Glu-Lys-Phe (SEQ ID NO: 11) 2010 + 360 12 Glu-Asp-Phe (SEQ ID NO: 12) 25 - 3 12 a Asp-Asp-Phe (SEQ ID NO: 13) 43 7 12b Phe-Asp-Asp-Phe (SEQ ID NO: 14) 55 - 14 13 Asp-Asp (SEQ ID NO: 15) 970 + 140 14 Asp-Glu (SEQ ID NO: 16) 5400 + 380 15 Asp-Gln (SEQ ID NO: 17) >1000 16 Glutamic acid ND 17 Aspartic acid ND 18 Phe-Leu-Arg-Gln-Asn-Leu-Ala-Phe (SEQ ID NO: 18) 2OOO Table 2. K. was obtained from a plot of the apparent K vs. I for peptides 1, 2 and 4 as shown in FIG. 2. For the others, K, was obtained from a plot of 1/V vs II at a saturating concentration of the substrate. The enzyme was assayed in the presence of the inhibitor in 50 mM sodium formate at pH 4.25 and 2.5 mM DTT. The final enzyme and substrate concentrations were 150 mM and 390 uM, respectively. Peptides 12a and 12b were assayed at pH 4.4 and contained 0.083% and 0.02% dimethylsulfoxide (“DMSO), respectively. “ND indicates no inhibition tested up to a concentration of 50 mM peptide. EDL has the smallest K, which is similar to the hexapeptide (peptide 2) and its analog EDF (peptide 12). The rate of HIV-1 protease-catalyzed hydrolysis of Sub 45 Strate I measured upon initiating the reaction by addition of TABLE 3-continued the enzyme to inhibitor/Substrate I mixture was equal to the rate in which the reaction was initiated by the addition of PEPTIDE NaCl Concentration, M K. M substrate I to a pre-incubated mixture (10 min) of enzyme so 2 1001 - 170 and inhibitor. Thus, the binding of these inhibitors was rapid Glu-Asp-Phe O 15 - 2 and reached equilibrium with the enzyme in less than 15 (SEQ ID NO: 12) 1. 410 - 25 Seconds Asp-Leu-Ala-Phe O 27O 25 (SEQ ID NO: 6) 1. 326O 390 EXAMPLE 3 Table 3. K. was obtained from a plot of 1/V vs II at saturating concentration 55 of the substrate. The enzyme was assayed in the presence of the inhibitor in CHARACTERISTICS OF TE INHIBITORS 50 mM sodium formate at pH 4.25 and 2.5 mM DTT. The final enzyme and substrate concentration were 150 mM and 390 uM respectively. Binding TF peptide inhibitors to the enzyme is dependent These results indicated that the interactions between the on an ionizable group in its protonated form with a pKa of enzyme and inhibitor appear to be mostly hydrophilic in 3.8 (FIG. 3). The pH rate profile for HIV protease-catalyzed 60 nature, i.e., electroStatic and hydrogen bonding, even in the hydrolysis of peptide Substrates showed pK values of presence of the one hydrophobic residue is the case in 3.4-3.7 for the basic group and 5.5 to 6.5 for the acidic peptide 12, Glu-Asp-Phe (SEQ ID NO: 12). The results also group. The observed pH dependence Suggested that the indicate that the observed increase in K at OM NaCl for the tripeptide inhibitor, EDL (SEQ ID NO: 4) interacts with the tripeptide analog, Glu-Asp-Phe (SEQ ID NO: 12), is not due catalytic aspartic acid residue at the active Site. Increasing 65 an increase in the hydrophobic affinity to bind to the active NaCl concentration decreased the affinity of the inhibitors Site cavity of the enzyme as a result of the Substitution of a Glu-Asp-Leu (SEQ ID NO: 4) and Asp-Leu-Ala-Phe (SEQ Phe in the place of Leu at the C-terminus. 5,872,210 15 16 EXAMPLE 4 serum and 150 ul of 10% Protein A Sepharose for one hour. The Sample was centrifuged at 12K rpm for two minutes. GLU-ASP-LEU (SEQ ID NO: 4) ISA One ml of the Supernatant was incubated with 10 ul of HIV-1 COMPETITIVE INHIBITOR FOR THE antibody positive human serum and 150 ul of Protein A MAMMALIAN ASPARTIC ACID PROTEASE, Sepharose for two hours at room temperature. The Samples PEPSIN were centrifuged and the Sepharose pellet was washed three Using the procedures described in Example 2, the K, times with PBS-TD buffer. The pellet was boiled for two value for the inhibition of pepsin by Glu-Asp-Leu (SEQ ID minutes with 100 ul buffer containing 2% SDS, 2% NO: 4) was found to be about 150 fold higher (7.5 mM at 2-mercaptoethanol, 10 mM Tris-HCl pH 6.8 and 15% glyc pH 3.0) than that for the inhibition of HIV protease. Thus, erol. The Sample was centrifuged and the labeled proteins in HIV protease was more selectively inhibited by EDL and its the Supernatant were separated in a 10% SDS-PAGE gel, analogs in the presence of other mammalian proteases. Such treated with Amplify dried and exposed to X-ray film. Selective inhibition facilitates treatment of HIV infected TF peptide inhibitors, EDL (SEQ ID NO: 4) or FLRED mammalian cells. LAF (SEQ ID NO: 1) were prepared in 200 mM or “blank” The inhibition of pepsin is notable. Catalytically inert 15 (no inhibitor) stock solutions. 1/25 to 1/200 dilutions were porcine pepsinogen is converted to enzymatically active added to infected cultures in 1-5 ml. Final concentrations of pepsin by loss of the first 44 N-terminal residues (Dunn, B. TF peptide inhibitors were 1-8 mM. M., et al. (1978), J. Biol. Chem. 253:7269–7276). The EDL, at a concentration of 2 mM resulted in a reduction precursor is activated to pepsinogen by an intramolecular of Gag processing by 50% in cell-extracted Samples and a cleavage event. Sequence analysis of the “pro” segment reduction of 70% in Supernatant Samples, as determined by amino acids reveals the porcine pepsinogen Sequence: Leu autoradiography of the p24 band. Arg-Gln-ASn-Leu at residues 12–16. Comparison of this sequence with the TF peptide inhibitors underscores the EXAMPLE 6 importance of the acidic residues (Glu and Asp) in the TF 25 peptide octomer Sequence: Phe-Leu-Arg-Glu-Asp-Leu-Ala USE OF TE INHIBITOR PEPTIDE TO SCREEN Phe (SEQ ID NO: 1). Substitution of the acidic residues (Glu FOR INHIBITORS OF HIV PROTEASE and Asp) with the neutral residues Gln and ASn results in a HIV protease is prepared as described, and complexed dramatic loss of inhibitory activity (see Table 2): Phe-Leu with labelled ("tagged”.) TF peptide or peptide cocktail, most Arg-Gln-Asn-Leu-Ala-Phe (SEQ ID NO: 18). preferably containing Glu-Asp-Leu (SEQ ID NO: 4) three Thus, some of the TF inhibitor peptides may have some amino acid inhibitor peptide. The complex is prepared under general inhibitory activity against mammalian proteases, the same conditions used for the protease activity assay Such as pepsin. described above. Equimolar amounts of a labelled TF pep tide and enzyme are used. Wells of 96-well microtiter plates EXAMPLE 5 35 are coated with the protease-TF inhibitor peptide(s) com INHIBITION OF VIRUS REPLICATION plex. The wells are then washed three times with phosphate buffered saline (PBS). Samples of the PBS wash are taken Cultures of Molt-3 and CEM cells which are CD4+ cell and assayed for the presence of unbound, labelled protease lines that are susceptible to HIV infection were chronically TF peptide complex. infected with a mixed population of HIV-1 virus isolates or 40 HIV-1 IIIB. Cells were infected at a density of 1–2x10 A test compound is then added to the Wells. One or more cells/ml in 4 ml of RPMI 1640 (supplemented with 10% concentrations of the test compound are typically used fetal bovine serum (FBS), 1% HEPES, 1% penicillin, 1% because the test compound is of unknown activity. Typically, Streptomycin). Infected cells were incubated with the appro concentrations are in 10-fold dilutions ranging from 10ng to priate concentration of the HIV-1 protease inhibitor for 2 45 0.1 mg concentrations. The 96-well format of the test kit hours in complete medium (RPMI 1640 with 10 percent makes Such dilution testing convenient and reduces the rate fetal calfserum) at 37°C. The cells (1x10 per ml) were then of both false positive “hits” and false negatives. The bound centrifuged and resuspended in methionine-free medium complex-test inhibitor mixture is incubated for 1 hour to with 1 percent dialyzed fetal calf serum and incubated for 30 overnight at room temperature 37 C. The temperature and minutes after which S-methionine was added to the cul 50 period of incubation may vary depending on the length and tures to a final concentration of 100 uC per ml. The cells charge of the particular peptides in the cocktail-complex and were labeled for 30 minutes and 1 ml of the culture was with the properties of a particular test compound. Aliquots centrifuged and saved for further analysis. After 30 minute of 10 ul are taken from the wells at time points after addition pulse labeling as above, the label was chased by the addition of the test compound to the well. The aliquot is assayed for of excess (100 fold) unlabeled methionine to the remaining 55 the presence of displaced, "tagged” TF peptide by described cultures. One milliter was withdrawn after 1, 2 and 4 hours methods that are dependent on the label. from the culture and centrifuged to Separate the cells and the In this way, any complex that is released following medium. The cell pellet was solubilized by the addition of 1 addition of the test compound is distinguished from dis ml of the PBS-TD buffer (PBS containing 0.5% Triton placed "tagged” TF inhibitor peptide. Appropriate controls X-100 and 1% deoxycholate). After 10 minutes in ice the 60 are used in which a “negative', compound-leSS buffer is Sample was centrifuged at 12000 rpm in an Eppendorf added to the well, and a “positive’, unlabelled TF peptide is centrifuge and the Supernatant was used for radioimmuno added to the well. Release of labelled TF inhibitor peptide precipitation assay (“RIPA). The labeled cell-free medium (above background controls) following addition of the test was solubilized by the addition of 2x PBS-TDS buffer. One compound indicates a high affinity of the test compound for ml of the Solubilized medium and 0.5 ml the cell extract was 65 the protease. Compounds discovered by this Screen may made to a final volume of 1 ml with PBS-TD buffer and then be tested directly for anti-protease activity as described incubated at room temperature with 10 ul of normal human in the examples above. 5,872,210 17 18 EXAMPLE 7 supplemented with 0.1 mM to 100 mM of TF peptide inhibitor. Conditions may vary somewhat for different pep ANTI-TF INHIBITOR ANTIBODIES tides due to different charge and length. Each preparation is Peptides were prepared at 1 mg/ml having the Sequence: Subjected to a pilot analysis in order to optimize Stabilization Cys-Gly-Gly-Gly-Gly-Gly-Phe-Leu-Arg-Glu-Asp-Leu 5 conditions. Virus titer protease activity is measured as Ala-Phe-OH described at 0° C. and 37 C. in samples containing the TF and peptide and in Samples not containing the TF peptide. Cys-Gly-Gly-Gly-Gly-Gly-Glu-Asp-Leu-Ala-Phe-OH. The loss of virus activity when the virus preparation Pre-bled NZW rabbits were inoculated intradermally with involves a live virus, Such as in a live, attenuated virus a mixture of 1.0 ml of peptide (500 ug) and 1.0 ml of vaccine, is measured as a loSS of Virus titer of about 0.3 log Freunds complete adjuvant (FCA) and subcutaneously with 10 after two days and 0.7 log 10 after 1 week at 37° C. The a mixture of 0.5 to 1.0 ml of peptide (250 ug) and 1.0 ml of attenuated virus Vaccine contains liquid or lyophilized virus, Freunds incomplete adjuvant (FIA). Two rabbits were inocu partially hydrolyzed gelatin, alcohol, cell culture medium, lated with each peptide preparation. The rabbits were acceptable buffer to proper pH and either Supplemented or boosted at approximately one month intervals for 3-6 15 devoid of TF peptides. months with test bleed Samples taken at each inoculation. Production bleeds, antibody preparation and analysis are EXAMPLE 10 performed as described (Current Protocols in Molecular STRUCTURAL ANALYSIS USING TF PEPTIDE Biology, Ausubel, F. M., ed John Wiley & Sons, Inc, 1994). INHIBITOR EXAMPLE 8 ATF peptide inhibitor is used to form crystals in complex with the HIV protease. Such crystal complexes allow for USE OF ANTI-TFPANTIBODIES TO INHIBIT detailed three dimensional Structural Studies using X-ray PROTEASE ACTIVITY crystallography and nuclear magnetic resonance (“NMR) Anti-TFP antibodies generated against the TF inhibitor 25 studies critical for the rational design of novel inhibitors of peptides are used to inhibit HIV protease activity in HIV polyprotein maturation. infected cells. Anti-TFP antibodies are incubated directly in Wild-type HIV-1 protease undergoes autolysis in 2 hours Virus-infected cultures of Susceptible cells including primary at room temperature, resulting in the loSS of enzymatic blood mononuclear cells (PBMNC) primary cells, and trans activity even at very low protein concentrations (1–3 uM). formed cell lines Such as H9, Molt-3, CEM, HeLa-CD4, and This has hampered biophysical Studies of the enzyme in the U-937 cells. The cells are grown in 0.2 to 5.0 ml or more of absence of inhibitors or in complex with Substrate analogs RPMI 1640 (supplemented with 10% fetal bovine serum with high K. To enable such analysis (including NMR and (FBS), 1% HEPES, 1% penicillin, 1% streptomycin) in a X-ray crystallography), a fully active protease variant was tissue culture flask and incubated at 37 C. The cells are isolated that contains Substitution mutations at potential Sites infected at an optimal cell concentration of about 1-2x10 35 of autolysis in the protease (A. Mildner et al. (1994), J. Biol. cells per ml. HIV-1 virus isolates are used to infect the cells Chem. 33,9405). This mutant protease which is kinetically at a multiplicity of infection (MOI) of 0.001 to 0.10, indistinguishable from the mature protease was incubated at followed by incubation at 37° C. Anti-TFP antibodies are high protein concentrations (300–330 uM) for up to 5 days added to the media either directly before, concurrent with, or at room temperature without any loss of catalytic activity. immediately after virus infection. Conditions vary for anti 40 Biophysical and kinetic analyses were performed as bodies raised against different peptides due to the different described (see Wondrak, E. M., et al. (1994), J. Biol. Chem. lengths and charges of the inhibitory peptides. Both positive 269:21948-21950; and Wondrak, E. M., et al. (submitted and negative controls are always performed, wherein Some 1995), J. Biol. Chem.) and indicated that substitution of the cultures are infected with "mock’ Solution in which no virus native Sequences flanking the N-terminus of the protease is present. Other cultures receive treatment of a Solution in 45 with non-native Sequences decreases the dimer Stability of which no antibody is present. Virus activity is monitored the protease. The purified fusion protein, containing 28 over time by reverse transcriptase (RT) assay and P24 assay non-native amino acids fused to the N-terminus of the as described Kageyama, S. et al. (1994), AIDS Res. and protease showed a K of 200-250 mM. This value is 40 Human Retroviruses, 10:735-745, in order to show inhibi times greater than that of the mature protease. An increase tion of virus activity by anti-TF antibody. 50 in the length of Sequence flanking the protease also decreases the conformational Stability of the protease. EXAMPLE 9 A 3-10 fold molar excess of the TFP to HIV-1 protease STABILIZATION OF VIRUS USING TF enzyme complex is prepared at 6 mg/ml in a buffer con 55 taining: (no salt, 0.1M Na Hepes (pH 7.5), 1.6 Na, K INHIBITOR PEPTIDE(S) Phosphate) or (No salt, 0.1 Na Acetate (pH 4.6), 2.0M Na The TF peptides are used to Stabilize a virus preparation Formate) or (No Salt, 0.1M Tris HCl (pH 8.5), 2.0M NH by inhibiting protease maturation, protease activity and virus Phosphate) or (30% w/v polyethylene glycol (“PEG”) autocatalysis. monomethylether 2000, 0.1M Sodium Acetate (pH 4.6), TF inhibitory peptides are added to a virus preparation 60 0.2M Ammonium sulfate). See Gorman, M.A., et al. (1992), including that of a live vaccine. J. Mol. Biol., 228:991–994; Erickson, J., et al. (1990), Susceptible cells are infected with HIV-1 as described Science, 249:527–533; Abdel-Megiud, S. S., et al. (1993), above. Infected cultures are monitored by RT assay for peak Biochem, 32:7972-7980. activity. Infected cells are centrifuged at 4 C. for 10 minutes The complex is prepared, crystalized, and analyzed as at 5,000 rpm and Supernatants are collected. Virus is purified 65 described (Tong, L., et al. (1993), Proc. Natl. Acad. Sci. USA by ultracentrifugation through a Sucrose gradient. Purified 90:8387–8391; Harrison, R. W., and Weber, I. T. (1994), Virus is resuspended in an appropriated buffer or in water Protein Engineering 7:1353-1363). X-ray diffraction and 5,872,210 19 20 NMR analyses are performed in order to elucidate the Viral protease catalyzes the hydrolysis of the Synthetic interactions between the TF peptide inhibitor and its analogs peptide spanning the TFP/TFp6 junction sequence. The and the HIV protease, thereby enabling the design of novel k/K, for the hydrolysis of the Substrate corresponding to potent inhibitors. the TFP/TFp6 cleavage site is quite similar to the hydrolysis of a Substrate spanning the TFp6/protease cleavage site (see EXAMPLE 11 Table 1). A polyprotein composed of the p7 and p1 domains of the Gag precursor is cleaved by the protease between p7 INHIBITION OF HIV-1-MEDIATED and p1 at about the same efficiency as that of the Substrate HYDROLYSIS OF SYNTHETIC SUBSTRATES encompassing the TFP/TFp6 cleavage site (Wondrak et. al. BY TF PEPTIDE INHIBITOR(S) (1993), FEBS lett. 333:21–24). But, in the Gag-Pol polyprotein, the p7/TFP junction is different in its sequence Reactions were performed in a total volume of 10 ul in 50 in the carboxyl side P3' and P4" position, amino acids, mM sodium acetate, pH 5.0 or 100 mM sodium phosphate involved in Substrate Specificity. A Synthetic peptide Span at pH 5.5 containing 1 mM DTT, 1 mM EDTA, 1M NaCl, ning this p7/TFP cleavage site, Glu-Arg-Gln-Ala-Asn-Phe in the presence of test substrate and 0.7 to 0.8 uM mature Leu-Arg-Glu-Asp (Substrate III), was hydrolysed far less HIV-1 protease and incubated at 37 C. Reactions were 15 efficiently than the Substrate corresponding to the TFP/TFp6 terminated with 190 ul of a solution of 8M guanidine cleavage Site, Asp-Leu-Ala-Phe-Leu-Gln-Gly-LyS hydrochloride and 1% trifluroacetic acid (TFA) and Sub (substrate IV) (See Table 1). The products Phe-Leu-Arg jected to reverse-phase high performance liquid chromotog Glu-Asp and Asp-Leu-Ala-Phe derived by the hydrolysis of raphy (“RP-HPLC) on a Cls column. The substrate and substrates III and IV, respectively, were isolated by cleavage products were separated with a linear gradient of 0 RP-HPLC and confirmed by electrospray mass spectrometry to 50% acetonitrile in water containing 0.05% TFA. The (M/Z=695.4 and 465.2 respectively). Provided that the identity of the cleavage products was confirmed by electro p7/TFP site is cleaved during virus maturation, TF peptides Spray mass spectroScopy. Peak areas were integrated using in principle represent P4'-P4 (Glu-Asp) fusion of two half the Turbochrom 4 software (PerkinElmer, San Jose, Calif.). substrates and will be the smaller of the proteolytic products Line weaver-Burk analysis was used to determine k and 25 of the Gag-Pol polyprotein in comparison to the Gag derived K. p1 and p2 proteins. Peptide substrates III and IV show It is significant to note that the TF region is flanked by inhibition of protease catalyzed hydrolysis of these sub Sequences that form Substrates for the viral protease. The Strates at lower pHS.

SEQUENCE LISTING

( 1) GENERAL INFORMATION: ( i i i ) NUMBER OF SEQUENCES: 19

( 2) INFORMATION FOR SEQ ID NO: 1: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 (B) TYPE: Amino Acid ( C ) STRANDEDNESS: Single (D) TOPOLOGY: Linear ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO: 1: P he Le u Arg G 1 u A s p Le u A a Phe 1. 5

( 2) INFORMATION FOR SEQ ID NO: 2: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: Amino Acid ( C ) STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

G 1 y G 1 u A s p Le u A 1 a P he 1. 5

( 2) INFORMATION FOR SEQ ID NO:3: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 4 (B) TYPE: Amino Acid ( C ) STRANDEDNESS: Unknown (D) TOPOLOGY: Linear 5,872,210 21 22 -continued

( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:3: G 1 u A s p Le u A 1 a 1.

( 2) INFORMATION FOR SEQ ID NO: 4: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 4: G 1 u A s p Le u 1.

( 2) INFORMATION FOR SEQ ID NO: 5: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 5: A rig G 1 u As p 1.

( 2) INFORMATION FOR SEQ ID NO: 6: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 6: A s p Le u A 1 a Phe 1.

( 2) INFORMATION FOR SEQ ID NO: 7: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 7: A s p G 1 u Le u 1.

( 2) INFORMATION FOR SEQ ID NO:8: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:8:

G u G u L. eu 1.

( 2) INFORMATION FOR SEQ ID NO: 9: ( i) SEQUENCE CHARACTERISTICS: 5,872,210 23 24 -continued (A) LENGTH: 3 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 9: A s p A s p L. eu 1.

( 2) INFORMATION FOR SEQ ID NO: 10: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

G I u. As in Le u 1.

( 2) INFORMATION FOR SEQ ID NO: 11: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 11: G 1 u Lys P he 1.

( 2) INFORMATION FOR SEQ ID NO: 12: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 12: G 1 u A s p P he 1.

( 2) INFORMATION FOR SEQ ID NO: 13: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 13: A s p A s p P he 1.

( 2) INFORMATION FOR SEQ ID NO: 14: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 4 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Ph e A s p A s p P he 5,872,210 25 26 -continued

( 2) INFORMATION FOR SEQ ID NO:15: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:15: A s p As p 1.

( 2) INFORMATION FOR SEQ ID NO: 16: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

( 2) INFORMATION FOR SEQ ID NO: 17: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

( 2) INFORMATION FOR SEQ ID NO: 18: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 18: P he Le u Arg G | n As n Le u A a Phe 1. 5

( 2) INFORMATION FOR SEQ ID NO: 19: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 (B) TYPE: Amino Acid (C)STRANDEDNESS: Unknown (D) TOPOLOGY: Linear ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 19: A rig G 1 in A a As in Phe Le u Arg G 1 u A s p Le u A 1 a Ph e Le u 1. 5 1 O

We claim: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID 1. A peptide consisting of at least one of SEQ ID NO: 1, NO: 16, or SEQ ID NO: 17. SEQID NO: 2, SEQ ID NO:3, SEQID NO: 4, SEQID NO: as 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID 2. A method of Screening a test compound for antiretro NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, Viral activity comprising: 5,872,210 27 28 contacting Said test compound with a retroviral protease 7. The test kit of claim 5 further comprising: complexed with a peptide of claim 1; and a negative control Sample in which no protease or inactive detecting displacement of Said peptide with Said com- protease is present, and ound; and assayingp Said compound for inhibition of protease activ 5 a positive control Sample in which protease with known itv. activity is present. 3. f method of claim 2 wherein said peptide is labelled. 8. A method of Stabilizing a retroviral preparation com 4. The method of claim 2 wherein the viral protease is prising the step of Supplementing the retroviral preparation immobilized onto a Solid matrix. with at least one peptide of claim 1. 5. A test kit for Screening a test compound inhibitory of 9. An antibody reactive with at least one peptide of claim aspartyl proteases comprising: 1. a. a peptide according to claim 1, and 10. An anti-idiotypic antibody reactive with the antibody b. an aspartyl protease. of claim 9. 6. The test kit of claim 5 further comprising a solid Support. k . . . .